Elsevier

Free Radical Biology and Medicine

Volume 63, October 2013, Pages 291-303
Free Radical Biology and Medicine

Original Contribution
N-Acetylcysteine and allopurinol up-regulated the Jak/STAT3 and PI3K/Akt pathways via adiponectin and attenuated myocardial postischemic injury in diabetes

https://doi.org/10.1016/j.freeradbiomed.2013.05.043Get rights and content

Highlights

  • Diabetic rats at early and late stages of the disease are less tolerable to ischemic insult.

  • N-Acetylcysteine and allopurinol confer synergistic cardioprotection via PI3K/Akt and Jak2/STAT3 pathways.

  • Cross talk between PI3K/Akt and Jak2/STAT3 pathways exists in diabetic myocardium.

  • Adiponetin serve as an mediator in N-acetylcysteine and allopurinol-mediated postischemic cardioprotection.

Abstract

N-Acetylcysteine (NAC) and allopurinol (ALP) synergistically reduce myocardial ischemia reperfusion (MI/R) injury in diabetes. However, the mechanism is unclear. We postulated that NAC and ALP attenuated diabetic MI/R injury by up-regulating phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathways subsequent to adiponectin (APN) activation. Control (C) or streptozotocin-induced diabetic rats (D) were untreated or treated with NAC and ALP followed by MI/R. D rats displayed larger infarct size accompanied by decreased phosphorylation of Akt, STAT3 and decreased cardiac nitric oxide (NO) and APN levels. NAC and ALP decreased MI/R injury in D rats, enhanced phosphorylation of Akt and STAT3, and increased NO and APN. High glucose and hypoxia/reoxygenation exposure induced cell death and Akt and STAT3 inactivation in cultured cardiomyocytes, which were prevented by NAC and ALP. The PI3K inhibitor wortmannin and Jak2 inhibitor AG490 abolished the protection of NAC and ALP. Similarly, APN restored posthypoxic Akt and STAT3 activation and decreased cell death in cardiomyocytes. Gene silencing with AdipoR2 siRNA or STAT3 siRNA but not AdipoR1 siRNA abolished the protection of NAC and ALP. In conclusion, NAC and ALP prevented diabetic MI/R injury through PI3K/Akt and Jak2/STAT3 and cardiac APN may serve as a mediator via AdipoR2 in this process.

Introduction

Myocardial infarction is a major cause of sudden death and is also one of the most common perioperative complications particularly prevalent in diabetes mellitus. Diabetes-induced oxidative stress has been suggested to be the major mechanism contributing to the development and progression of myocardial infarction [1], [2]. Restoring blood flow to the ischemic heart is clearly necessary for myocardial salvage. However, reperfusion can further exacerbate myocardial ischemia/reperfusion (MI/R) injury due to aggravated oxidative stress injury. Therefore, intensive research has been focused on the various pathophysiological mechanisms related to MI/R injury and on the development of potential therapeutic strategies. Our previous study found that antioxidants N-acetylcysteine (NAC) and allopurinol (ALP) can confer synergy in combating MI/R injury in diabetic rats [3]. However, the underlying mechanism is unclear.

Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) represents one of the most important defense mechanisms against MI/R injury. Activation of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway plays critical roles in the activation of eNOS and the subsequent attenuation of MI/R injury [4], [5]. However, NO is decreased in diabetic myocardium [6], [7], potentially due to reduced Akt and STAT3 activation and subsequent reduction of eNOS activation. This makes the diabetic heart more sensitive to MI/R [8], [9]. Thus, strategies that can activate Akt and STAT3 in diabetes may also be effective in attenuating MI/R injury. The antioxidant compound H-2693, a modified mexiletine derivative, can attenuate MI/R injury by inducing Akt activation [10]. Similarly, NAC and ALP can restore Akt and STAT3 activation before MI/R in the myocardium of diabetic rats and this was associated with significant attenuation of postischemic myocardial infarction [3]. It is plausible that activation of Akt and STAT3 may represent the major mechanism by which NAC and ALP confer synergy in attenuating MI/R injury in diabetes.

Oxidative stress can down-regulate adiponectin (APN), an adipose-specific plasma protein that possesses insulin-sensitizing, antiapoptotic and anti-inflammatory properties [11], [12]. Plasma APN levels are reduced in diabetic patients [13] and STZ-induced diabetic rats [3], which may be the major factor that has rendered the diabetic heart less resistant to ischemic insult given that APN deficiency can lead to increased myocardial damage in response to ischemic insult while APN supplementation increased NO production and attenuated MI/R injury [14], [15], [16]. Also, oxidative stress decrease APN production and reduces the activation of both Akt and JAK/STAT, two important signaling pathways involved in MI/R injury [17]. These investigations collectively suggest that antioxidant treatment may activate both Akt and JAK/STAT, potentially in part via APN activation, and attenuate MI/R injury in diabetes.

We have shown that NAC and ALP confer a synergistic effect on restoration of cardiac APN content and APN receptor 2 (AdipoR2) expression with concomitant increase in eNOS activation before MI/R insult, which could be a potential mechanism for enhancing myocardial resistance to MI/R injury [3]. However, since MI/R is the major cause of increased oxidative stress which can cause disruptions in cellular signaling and contribute to tissue injury, whether or not antioxidants can change the postischemic APN content and APN-related signaling pathways as well as the underlying mechanism remains unclear. Therefore, in the present study, we focused on the postischemic pathophysiological changes and using PI3K/Akt or Jak2/STAT3 inhibitors/siRNAs and/or APN adenovirus to test the hypothesis that NAC and ALP confer synergy in reducing MI/R injury in diabetes, primarily by activating PI3K/Akt and Jak2/STAT3 pathways and that APN activation plays a critical role in this process. This hypothesis was tested both in in vivo models of MI/R in STZ-induced diabetic rats and in vitro in cultured rat cardiomyocytes exposed to high glucose and subjected to hypoxia/reoxygenation (H/R).

Section snippets

Induction of diabetes

Male Sprague-Dawley rats (250±10 g) supplied by the Laboratory Animal Service Center (University of Hong Kong) were used. The experiments were performed after obtaining approval from the Committee on the Use of Live Animals in Teaching and Research (CULATR). Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ) (Sigma-Aldrich, St. Louis, MO) as described [3].

Experimental protocol

Rats were randomly divided into five groups:

  • Control (C)

  • Diabetes (D)

  • Diabetes treated with NAC and ALP

General characteristics and free 15-F2t-IsoP and APN levels before MI/R

As shown in Table 1, STZ-induced diabetic rats at early stage (5 weeks) displayed increased plasma glucose and increased water intake and food consumption (all P<0.05 vs control). NAC and ALP markedly reduced food consumption and water intake (P<0.05). Body weights were lower and plasma glucose was elevated in diabetic group as compared to control (P<0.05). NAC and ALP treatment had no significant impact on body weight or glucose level in diabetic rats. Plasma and cardiac-free 15-F2t-IsoP were

Discussion

There are several novel findings in the current study. First, using in vivo and in vitro models of MI/R, we demonstrated that the cardioprotective effects mediated by a combination of NAC and ALP were associated with enhancement of Akt, STAT3, and eNOS activation and an increase in NO production in response to MI/R insult. All these beneficial effects of NAC and ALP were attenuated by either the PI3K inhibitor wortmannin or the Jak2 inhibitor AG490. This suggests that activation of Akt and

Acknowledgments

This work was supported by Research Grants Council (RGC) of Hong Kong GRF grants (781109M, 766709M, 784011) and the National Natural Science Foundation of China (81200609). The authors are grateful to Dr. Xia Gao (Department of Endocrinology, Beijing Chaoyang Hospital Affiliated to Capital Medical University, Beijing, China) for her assistance in echocardiography in this study. Dr. Zhengyuan Xia takes full responsibility for the work as a whole, including the study design, access to data, and

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